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International Research Journal of Agricultural Science and Soil Science (ISSN: 2251-0044) Vol. 2(11) pp. 469-475, November, 2012
Available online http://www.interesjournals.org/IRJAS
Copyright ©2012 International Research Journals
Full Length Research Paper
Bioactivity of methanolic seed extract of Barringtonia
asiatica L. (Kurz) (Lecythidaceae) on biological
characters of Spodoptera litura (Fabricius)
(Lepidoptera: Noctuidae)
Danar Dono, Wahyu D. Natawigena and Mubqi Ghaida Majid
Department Plant Pests and Diseases, Faculty of Agriculture, Universitas Padjadjaran, Jatinangor 45363,
Indonesia
ABSTRACT
Plants are known to have various chemical compounds that have potential to be developed as
insecticides. One of the potential plants to be developed as a source of insecticides is Barringtonia
asiatica (Lecythidaceae). This research was conducted to determine toxicity of methanolic seed extract
of Barringtonia asiatica to mortality and biological character of Spodoptera litura. The evaluation of
toxicity was carried out using feeding method. Result of this research indicated that methanolic seed
extract of B. asiatica had insecticidal activity with LC50 at concentration of 0.30% and LC90 at
concentration of 0.80% in 13 days after treatment with LT 50 at 4.8 days. In addition, methanolic seed
extract of B. asiatica caused decrease of larval weight, tend to increase duration time of development,
reduced leaf consumption and decrease of egg amount oviposited by female of S. litura.
Keywords: Barringtonia asiatica, seed, extract, mortality, biological character, Spodoptera litura.
INTRODUCTION
Various active ingredients originally from plants have
been known and tested against insects. At least 2000
plant species have been reported toxic to various plant
pests, and more than 850 active compounds from plants
have been tested against insects (Grainge and Ahmed,
1988; Prakash and Rao, 1997). During the last decade
there is improvement of big enthusiasm in seeking of
insecticide compounds from plant (Schmutterer, 1995).
Syahputra (2001) reported from various districts in
Indonesia, there were more than 40 potential plant
species that could be used as botanical insecticides.
Botanical insecticides have long been applied by the
farmers. One of the plants having a potency to be
*Corresponding Author E-mail: [email protected]
developed as insecticide is Barringtonia asiatica Kurz
(Lecythidaceae) with common name sea poison tree or in
Indonesia known as bitung (Ecology and Evolutinonary
Biology Greenhouse (EEBG), 2006).
B. asiatica is known to have active compounds which
cause mortality to insect pests. Methanolic seed extract
of B. asiatica was toxic to Crocidolomia pavonana with
value of LC50 equal to 0.66% at 7 days after treatment
(dat). The application of seed extract of B. asiatica also
has influenced oviposition with effective concentration
equal to 0.96% which causes C. pavonana female not to
lay the eggs at the crop. Larval response indicated that
the extract of B. asiatica, besides had toxic character also
had antifeedant activity (Dono and Sujana, 2007).
Therefore, methanolic seed extract of B. asiatica had a
potency as insecticide. One of the active compounds in
seed of B. asiatica is saponin (Herltz et al., 2002; Burton
et al. 2003, Cannon et al. 2004). At some places B.
470 Int. Res. J. Agric. Sci. Soil Sci.
asiatica is used as ancient medicine and fish poison (The
Cook Island Nature Heritage Trust, 2005). Active
compound in seed of B. asiatica which poisoned fish is
group of saponin compounds (Tan, 2002; EEBG, 2006).
One of the most toxic compounds against fish from B.
asiatica seed extract is ranunkosida VIII (Burton et al,
2003). Research about seed extract of B. asiatica has
been conducted but its toxicity to Spodoptera
litura(Fabricius) (Lepidoptera: Noctuidae) has not been
known. This research was conducted to know the
influence of B. asiatica seed extract on mortality and
biological characters of S. litura.
S. litura is widespread insect, polyphagous, and can
eat all parts of crop, especially at dry season
(Department of Indonesian Agriculture, 2005). Controlling
of S. litura in general is still by using synthetic insecticide.
Unfortunately, usage of synthetic insecticides has
reported able to generate resistance of insect target,
resurgence, and secondary pest out break. Other side
impact of usage of synthetic insecticide is environmental
contamination and killing of non-target organism (Profit,
1993). Therefore, an alternative insecticide, that was
relatively safe for environment and has minimal or no
side effect to non target organism, was required. An
alternative method for controlling of the pest was
botanical insecticide from seed extract of B. asiatica.
MATERIALS AND METHODS
Rearing of Test Insect Spodoptera litura
Test insect larvae, S. litura, was collected from
Brassicaceae plantation in Lembang. Larvae obtained
from the field were placed in plastic box and feed with
taro leaf, and pupation took place in plastic box with
sterile soil. Pupae were put in screen cage and adults
emerged from pupae was fed with dilution of honey 10%
which have been permeated in cotton. In the cage, some
leaves and pieces of taro were placed for adult
oviposition. Egg mass at taro leaf was removed into
separate plastic box. At the time of egg hatching, larvae
ware moved to bigger plastic box and fed with taro leaf.
Extraction of Seed of Barringtonia asiatica
Seeds of B. asiatica was obtained from the field (altitude:
750 m above the sea level) of Campus Universitas
Padjadjaran, campus Jatinangor, Sumedang, West Java,
Indonesia. Seed was taken away from the fruit, then
sliced thin and wind-dried. The dried seeds were refined
by using blender to form flour. Flour of the seeds at
weight of 2969.34 g was soaked in 10.68 litres of
methanol. Methanol was used as solvent because of
active compound contain in seed of B. asiatica was polar
compound. The solution was filtered using filter paper
and evaporated using rotary evaporator at temperature of
40ºC with low pressure until all solvent evaporated and
yielded of 781.48 g crude extract. The crude extract was
stored in refrigerator at temperature ± 4ºC until being
used.
Experiment 1. Insecticidal Activity of B. asiatica
Against Spodoptera litura
The extract was tested against second instar larvae of S.
litura using a leaf residue feeding method. Extract was
tested at six concentrations that were expected to cause
between 0 – 100% insect mortality. The range of
concentrations was determined by preliminary test. The
treatment with each concentration was replicated six
times.
Examination was done with taro leaf dipping method.
The extract diluted with mixture of aquadestilata with
Agristic (0.05%). Two treated or control leaf disks were
placed in glass petri dishes (9 cm in diameter) lined with
towel paper, and 10 first instar S. litura were placed in
each dish. The test larvae were fed with treated leaves
for 72 hours, then fed untreated leaves until fourth instar.
Parameter observed was mortality of insect.
Regression relationship between extract concentration
and percentage of insect mortality were determined by
probit analysis (Finney, 1971). Calculations of sublethal
concentrations of extract were used for other
experiments.
Experiment 2. The Effect of B. asiatica Seed Extract
on Fecundity and Biological Character of Spodoptera
litura
The treatment procedures were similar with the same as
Experiment 1. Concentration of extract tested at this
research equivalent with LC30, LC50, and LC70 determined
at experiment 1. In each level concentration of test and
control larvae was observed until becoming adult. Imagos
that were successfully emerged from pupae were paired
and then put into different plastic cage (diameter 6.5 cm,
height 30 cm). The imago fed with diluted honey (30%)
and taro leaf put in plastic container as egg trap.
Observation was done every day for larval weight
using digital weighting-machine, feed wide eaten, pupa
weight, emerge of adult, number of eggs oviposited
during the female of adult lived. Larval weight was
measured by electrical weighing-machine. While width of
food leaf consumed was measured with paper millimeter
block. Data obtained were analysed with analysis of
variance and continued with multiple range test at
significance level of 5%.
Dono et al. 471
100
90
80
Control
0.05 %
0.1 %
0.2 %
0.4 %
0.8 %
Mortality (%)
70
60
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9
10
11
12
13
Day after treatment (dat)
Figure 1. Mortality of S. litura larvae caused by treatment of methanolic seed extract of B. asiatica
Table 1. Parameter regression probit relationship between concentration of methanolic seed extract of B. asiatica and mortality of S. litura
Day analisys
(DA)
3
5
8
10
12
13
a
b
SE
g
a ± SE
-0.55 ± 0.26
-0.82 ± 0.24
0.32 ± 0.23
0.51 ± 0.23
1.18 ± 0.28
1.12 ± 0.26
b ± SE
Value of
LC50
0.71 ± 0.38
1.08 ± 0.38
1.27 ± 0.35
1.46 ± 0.36
2.37 ± 0.49
2.15 ± 0.46
(%)
5.89
1.18
0.55
0.44
0.31
0.30
Confidential
limits (95%)
Value of
LC90
Confidential
limits (95%)
0.56 – 27.33
0.33 – 1.57
0.29 – 0.88
0.22 – 0.43
0.21 – 0.41
(%)
363. 53
17.99
5.57
3.31
1.09
1.17
3.22 – 47.42
1.82 – 196.3
1.39 – 34.35
0.71 – 2.72
0.74 – 3.05
g
1.01
0.29
0.29
0.23
0.16
0.15
= Intercept
= Coefisien of regression
= Standard error
= Potential index of significance
RESULTS AND DISCUSSION
Toxicity of Methanolic Seed Extract of Barringtonia
asiatica to Spodoptera litura Larvae
Mortality of test insect increased equivalently with
increasing concentration of methanolic seed extract of B.
asiatica applied. In all treatment, larval mortality was
seen since 1 day after treatment (dat). Improvement of
mortality at concentration of 0.05%, 0.1%, and 0.2% was
seen stable at 6 dat, while at concentration of 0.4% and
0.8%, improvement of mortality always happened until 11
dat (Figure 1).
Increase of test larval mortality was seen in the first
day until 6 DAT. Increasing of test larval mortality after
seventh DAT was not significant and at low concentration
the larval mortality tend to be stable. The result of
research conducted by Dono and Sujana (2007)
indicated that methanolic seed extract of B. asiatica
tested to Crocidolomia pavonana cause death and inhibit
the development of larvae.
Bioactivity of active compound in methanolic seed
extract of B. asiatica worked slowly with mortality
response equal to 90% at concentration of 0.8% at 13
DAT. Saponin is principal active compound in extract of
B. asiatica acted as stomach poison so that it doesn't
cause death of larva immediately (Agrell et al. 2004).
Methanolic seed extract of B. asiatica has insecticidal
activity to S. litura larvae. Based on result of lethal
concentration (LC50) analysis, the value of LC50 since 3
DATuntil 5 DATwere decreasing and stable at 12 DAT.
Value of LC50 obtained at 13 DATwas equivalent with
concentration of 0.30% (Table 1).
Amount of larval mortality generally occurred at initial
instar compared late instar. Difference of level of defence
in detoxyfication of poison compound in body causing
472 Int. Res. J. Agric. Sci. Soil Sci.
Table 2. Analysis LT50 methanolic seed extract of B. asiatica to S. litura larvae
Extract
concentration (%)
0.2
0.4
0.8
a ± SE
-1.25 ± 0.31
-1.09 ± 0.29
-1.02 ± 0.29
Value of LT50 (days)
b ± SE
1.01 ± 0.34
1.11 ± 0.33
1.74 ± 0.34
Confidential
limits (95%)
10.02 – 27.07
6.22 – 20.64
3.36 – 4.46
16.9
9.5
4.8
g
0.42
0.33
0.14
a = Intercept
b = Coefficient of regression
SE = Standard error
g = Potential index of significance
LT = Lethal time
Weight of larvae (g)
0.3
0.25
0.2
Control
0.15 %
0.30 %
0.52 %
0.15
0.1
0.05
0
5
6
7
8
9
10
11
12
13
14
15
Day after treatment (dat)
Figure 2. Average of weight of S. litura larvae on each methanolic seed extract of B. asiatica
treatment at concentration equivalent with LC30 (0.15%), LC50 (0.30%), and LC70 (0.52%)
death response will differ in. According to Syahputra et
al. (2006), if larva consumes active compoundm initial
instar of larva more sensitive to toxic compound and
would easily to die compared to late instar of larva
because of the late instar has stronger defence reaction
compared to initial instar.
Death symptom of larvae showed that initially the
larvae were not active,the body looked smaller, seen
wrinkled, and then dry. This was indicated that active
compound in the form of saponin contain in methanolic
seed extract of B. asiatica wasthe main cause of death of
larva at all of treatment. According to Chaieb et al.
(2001) saponins in the insect body can disturb cell
permeability and cause decreased size of the cell
because of losing of cell fluid. The breakdown this cell
permeability is caused by the reaction between saponins
with cholesterol and phospolipid of the cell membrane
(Menger and Keiper, 1998).
B. asiatica seed extract at lower concentration of
extract required more time to cause mortality of test
insect compared with height concentration. B. asiatica
seed extract at concentration of 0.8% was killing 50% of
insect test slowly (LT50 equivalent with 4.8 days), while at
concentration of 0.2%, value of LT50 far longer that was
16.9 days (Table 2). Saponin extracted from plant cause
death of test insect sufficiently long (Agrell et al. 2004).
Influence of Methanolic Seed Extract of B. asiatica to
Biological Character of Spodoptera litura Larvae
Influence of Methanolic Seed Extract of B. asiatica on
Weight of Larvae
Larval weight was depended on quantum of food
consumed. Decrease in feeding activity caused as a
result of toxic compound eaten by larval test can
influence growth rate and larval weight (Ambarningrum et
al., 2009). This caused difference in improvement of
larval weight for every concentration of B. asiatica seed
extract tested. Improvement of average of larva weight at
control and concentration of 0.15% increasing so closing
0.3 g while at concentration of 0.30% and 0.52%,
improvement of average of larva weight still below
(under 0.2 g) (Figure 2).
Difference of average of larva weight in each
concentration showed that at height concentration of
methanolic seedextractof B. asiatica can reduce feeding
Dono et al. 473
Table 3. Influence of B. asiatica seed extracts to leaf area consumed by S. litura larvae
Extract concentration (%)
Leaf area consumed at 4
dat (%) (x ± SE)(N)
Control
0.15 (LC30)
Leaf area consumed at 2
dat (%) (x ± SE) (N)
2.40 ± 0.86 (60) b
1.88 ± 0.36 (60) b
0.30 (LC50)
1.87 ± 0.48 (60) b
12.26 ± 5.51 (40) b
0.52 (LC70)
0.93 ± 0.23 (60) a
5.05 ± 2.05 (38) a
20.75 ± 3.07(60) c
14.30 ± 6.79 (58) b
The mean followed by different letter was significance different (α = 0.05)
x
= average of leaf consumed (mm²)
SE = Standard error
N
= Number of alive larvae
dat = day after treatment
Table 4. The effect of methanolic seed extract of B. asiatica to developmental time of S. litura larvae (day)
Extract
Concentration
Control
0.15 %
0.30 %
0.52 %
Developmental Time
Instar I–II (day)
(x ± SE) (N)
2.0
2.0
2.0
2.0
± 0.00 (60)
± 0.00 (60)
± 0.00 (60)
± 0.00 (60)
Developmental Time
Instar II–III (day)
(x ± SE) (N)
1.7 ± 1.64 (60)
1.9 ± 0.40 (46)
2.0 ± 0.22 (33)
2.2 ± 0.44 (26)
Developmental Time
Instar III-IV (day)
(x ± SE) (N)
3.0 ± 0.74 (58)
3.7 ± 0.48 (44)
4.0 ± 0.25 (29)
4.0 ± 0.24 (21)
x = Mean of duration of larval development (days)
SE = Standard error
N = Amount of larvae alive
activity of S. litura, causing decreasing of average of
larval weight. Toxic compounds contained in seed extract
of B. asiatica can influence response of insect physiology
(Dadang and Prijono, 2008).
Influence of Methanolic Seed Extract of B. asiatica to
Leaf Area Consumed by Spodoptera litura Larvae
Leaf area consumed by the test larvae indicated smaller
with increase of the extract concentration (Table 3). The
extract (saponin as main active compound contained in
the methanolic seed extract of B. asiatica) indicated to
act as antifeedant. Result of Herlt et al. (2002) research
indicated that two main saponins isolated from B. asiatica
shown anifeedant activity against Epilachna. The same
results were indicated by some researchers (Adel et al.,
2000; Brown (2006); Szczepanik et al., 2001; Shinoda et
al., 2002; Agerbirk et al., 2003; Agrell et al., 2003;
Szczepanik et al., 2004).
Insect is a heterotroph which must consume other
organisms to obtain molecules rich in energy which is
needed to its growth and development (Meyer 2005).
When the nutrition is not enough, it showed disturbance
to metabolic process of the insect which would cause
death or abnormal growth and development process.
Influence of Methanolic Seed Extract of B. asiatica to
Duration of Larval Development
Methanolic seed extract of B. asiatica influenced
development time of larvae from instar II until instar IV. At
higher concentration of B. asiatica seed extract lengthen
duration of development time of the test larvae (Table 4).
When larvae consumes toxic active compound, some of
larvae will death, while another larvae will maximize
energy source in the body to eliminate the toxic
compound so that the growth and development retarded
(Syahputra et al., 2006; Tomia, 2008).
Influence of B. asiatica Seed Extract to Pupal Weight
and Emergence of S. litura adult
B. asiatica seed extract influenced pupal formation and
weight, and adult emergence (Table 5). Pupal formation
at treatment was smaller than control pupae. This
indicated that nutrition supplay annoyed at the larval
stage. The growth of larvae becomes pursued and finally
will influence development of S. litura larvae when
forming pupa. Forming of imperfect pupa can reduce
emergence of insect adult.
There was failure in pupal formation at treatment with
474 Int. Res. J. Agric. Sci. Soil Sci.
Table 5. Effect of B. asiatica seed extract on pupal forming and weight, adult emergence of S. litura
Treatment
Control
0.15 % (LC30)
Pupal forming (%)
39.65
39.53
Pupal Weight ± (SE) (g) (N)
0.28 ± 0.34 (20)
0.28 ± 0.23 (17)
Adult emergence (%)
0.69
0.53
0.30 % (LC50)
35.71
0.26 ± 0.21 (15)
0.52
0.52 % (LC70)
33.33
0.25 ± 0.17 (11)
0.45
SE = Standard Error
N = Number of insect test
g
= gram
Table 6. Amount of egg laid by adult of S. litura emerged from test
insect treated with methanolic seed extract of B. asiatica in larval period
Extract concentration (%)
Control
0.15 (LC30)
Amount of eggs (x ± SE)
1343± 0.22 a
1236± 0.12 a
0.30 (LC50)
1172 ± 0.42 a
0.52 (LC70)
1138 ± 0.15 a
x = Number of eggs reproduced
SE = Standard error
higher concentration of extract. It indicated the lack of
nutrition supply to S. litura larvae as resulted inhibition of
insect feeding activity by active compound of B. asiatica
seed extract. In addition, failure in pupal formation could
be due to decreasing of food consumption by the insect
as a result of toxic compound (Adel et al., 2000).
The treatment with concentration of 0.15%, the pupa
that was formed successfully, equal to 39.53% with
emergence of adult equal to 0.53%, while at
concentration of 0.52%, pupal formed only equal to
33.33% with emergence of adult only 0.45%. Pupa that
was not formed perfectly at concentration of0.52%
caused decreasing of adult emergence. The data
indicated that there was decreasing activity of methanolic
seed extract of B. asiatica which in line with decreasing of
extract concentration.
Influence of Methanolic Seed Extract of B. asiatica to
Fecundity of Spodoptera litura Female
Number of eggs yielded at all of treatments did not
different (Table 6). This indicated that active compound in
methanolic seed extract of B. asiatica did not caused
decrease of reproduction level of S. litura with indicated
that most of toxic compound that successfully enters into
larval body was successfully neutralized when caterpillar
grows from initial instar towards final instar in parallel with
moulting process of insect.
The value followed by the same letter not significantly
different (Duncan test at α= 0.05)
The findings above increasingly strengthens
estimation that active compound activity coming into
larval body of S.litura as a result of treatment of
methanolic seed extract of B. asiatica will decline and
disappear in line with growth and development from larval
stadium, pupa and then adult. Defence mechanism in
insect body will try to eliminate toxic compound coming
into the insect body and cause imago to successfully
emerge from pupa will return to normal growth and can
lay eggs again normally.
CONCLUSION
Result of this research indicated that methanolic seed
extract of B. asiatica had insecticidal activity with LC50 at
concentration of 0.30% and LC90 at concentration of
0.80% in 13 days after treatment with LT50 at 4.8 days.
In addition, Methanolic seed extract of B. asiatica coused
decrease of larva’s weight, time of development, reduced
leaf consumption
and decrease of
egg amount
oviposited by female of S. litura. Therefore, methanolic
seed extract of B. asiatica has potential to be developed
as an insecticide to control Spodoptera.
Dono et al. 475
ACKNOWLEDGEMENT
This research is a part of the research funded by the
Indonesian Ministry of Education and Culture under the
program of Riset Andalan Perguruan Tinggi dan Industri
(RAPID), 2011 to who we greatly indebted.
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